Vaccination consists in immunizing a person or an animal against an infectious disease, generally by administering a vaccine to a person or animal. Vaccines, which stimulate the immune system, protect the person or animal against an infection or a disease.
It is established that human vaccination thus makes it possible to combat and eliminate potentially deadly infectious diseases, and it is estimated that in this way more than 2 to 3 million deaths per year are prevented. It is one of the most profitable investments in the health field.
A vaccine is an antigenic preparation which makes it possible to induce, in the person or animal vaccinated, a pathogenic agent-specific immune response capable of protecting them against natural infection or of attenuating the consequences thereof.
The flu is a common viral respiratory infection, and seen throughout the world, which evolves in epidemic winter episodes in tempered regions, due to influenza viruses. The World Health Organization (WHO) considers that they cause between 3 and 5 million serious cases and 250 000 to 500 000 deaths per year worldwide (http://www.pasteur.fr/fr/institut-pasteur/presse/fiches-info/grippe#sthash.PwUNmJ10.dpuf). The influenza viruses responsible for pathological conditions in humans are the type A and B influenza viruses. While type B influenza viruses circulate in the lineage form, type A influenza viruses are categorized in viral subtypes according to the antigenic properties of the major two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Influenza viruses have between 300 and 800 glycoproteins at their surface associated with an NA/HA ratio that can range from one to ten.
The viruses circulating in humans and responsible for seasonal epidemics are the viruses A (H1N1) and A (H3N2). Since the main reservoir of influenza viruses is the animal reservoir (avian and porcine), animal viruses can cross the species barrier and infect humans. Viruses such as avian virus A (H5N1) and pandemic virus A (H1N1) can cause serious public health problems.
In point of fact, vaccination is for the moment the only effective means for protecting populations against influenza viruses. The “seasonal” vaccine makes it possible to acquire immunity against the seasonal circulating viruses A such as (H1N1) and (H3N2) and the various lineages of virus B. It is defined each year on the basis of the prototype strains of the prior year and contains the antigens of the type A and type B seasonal viruses. The host's immune response is mainly of humoral type with expression of neutralizing antibodies that are directed against the HA and NA proteins for the viruses.
Because of a considerable antigen shift in these two proteins, the vaccine composition must be re-evaluated annually.
The conventional process for producing a vaccine is based firstly on obtaining, by genetic reassortment between the A/PR8/34 (H1N1) strain and the seasonal virus strains, vaccine seeds on eggs, for each of the annual prototype A strains determined by the WHO. Most vaccine manufacturers usually use these reassortment viruses deriving mainly from the parental A/PR/8/34 virus. Thus, each vaccine seed is derived from a process of genetic reassortment between the prototype A strain and the A/PR8/34 (H1N1) virus which possesses optimal replicating capacities in eggs. The production of this type B virus is carried out directly on eggs without reassortment with PR8.
The viral particle contains eight distinct gene segments, consisting of a single RNA chain linked to nucleoproteins and associated with a polymerase complex, each of the genes encoding one to three given proteins of the virus: HA, NA, M1, M2, NP, NS1, NEP, PB1, PB1F2, PB1N40, PB2 and PA. Consequently, a long selection process makes it possible to obtain the vaccine reassortment, comprising at least the gene segments encoding HA and NA of the prototype strain on the genetic background of the PR8 virus (“6+2” composition). The 2 vaccine reassortants, resulting from the genetic reassortment between the 2 prototype strains and the parental A/PR/8/34 strain, are then amplified on eggs. The HA and NA antigens are extracted from allantoic productions, and optionally combined with adjuvants in order to produce the vaccine doses.
This industrial process for the production of vaccine doses is long (4 to 6 months). However, in the case of the production of pre-pandemic vaccines, in particular against human flu, it has been evaluated by the WHO that approximately 1 billion people would have to be vaccinated if a plan for combatting a flu pandemic was to be implemented. In point of fact, such a combatting plan will depend partly on the efficiency of the bulk and rapid production of vaccines.
In order to face up to the increasing demand for vaccines against the seasonal circulating strains, but also to the demand—that is difficult to predict—for vaccines against one (or more) potentially pandemic emerging strains, the availability of eggs may prove to be a limiting factor, all the more so since risks of avian pandemics in livestock persist. There is, however, a constant difficulty of producing a good yield of B strains in eggs, and also some type A reassortants which are not 6+2 but instead 5+3 or 4+4.
Thus, in order to reduce the time between the selection of the circulating prototype strains and the production of the vaccine doses, alternative strategies have been developed. Influenza viruses can also be produced in cell systems (Barrett P N, Portsmouth D, Ehrlich H J. Developing cell culture-derived pandemic vaccines. Curr Opin Mol Ther. 2010 February; 12(1):21-30 ([1]); Le Ru A, Jacob D, Transfiguracion J, Ansorge S, Henry O, Kamen A A. Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing. Vaccine. 2010 May 7; 28(21):3661-71 ([2])). The production of vaccine strains and also the amplifications thereof can be carried out on cells using industrial bioreactors. Indeed, the use of a cell line for amplifying vaccine strains makes it possible, inter alia, to no longer be dependent on the “egg” system (amount of egg and production yield insufficient for managing a pandemic), reduces the surface antigen modifications regularly observed in allantoic production and does not cause allergies. This strategy makes it possible to more readily meet the vaccine demand during the emergence of a pandemic influenza virus (for example A H1N1).
Nevertheless, these systems, and in particular the registered cell lines (MDCK, Vero, PERC6, EB66, etc.) do not allow optimal replication of influenza viruses, thereby constituting a major limiting factor in terms of vaccine seed production. Production yields in cell systems are still currently lower than those obtained in the allantoic system. Thus, at the current time, few manufacturers have chosen this new production method since the industrial process is far from being as effective as eggs for the moment.
In order to reduce as much as possible the vaccine dose production time and with the objective of having an equivalent yield level, in terms of number of doses, the obtaining of the vaccine seeds can be carried out by using reverse genetic techniques, making it possible to more rapidly obtain the vaccine reassortant of “6+2” composition, thus eliminating all the selection steps. The production of recombined viruses by reverse genetics provides the most realistic alternative for efficiently meeting vaccine demand. The production of recombined viruses by reverse genetics provides the possibility, secondly, of producing an “optimized” PR8 virus making it possible, when it is used by means of a process of genetic reassortment with prototype strains, to produce vaccine reassortants having optimized viral characteristics for in-egg or in-cell vaccine dose production. This type of method can also be applied for in-cell production.
Another strategy for optimizing vaccine production in allantoic and cell systems is aimed at increasing the yield of the viral production on the basis of genetic modifications of the vaccine seeds and/or using small chemical molecules of interest targeting the host cell. Such processes are undergoing evaluation (FR 10/55716 ([3]), WO 2012007380 ([4]), FR 10/59132 ([5]), WO 2012059696 ([6])).
Another strategy consists in improving the efficacy of the vaccine antigens, by adding adjuvants, thereby making it possible to produce more vaccine doses for the same limited number of eggs and the same amount of glycoproteins produced (Ellebedy A H, Webby R J. Influenza vaccines. Vaccine. 2009 Nov. 5; 27 Suppl 4:D65-8 ([7])). This process is more recommended for people with a deficient immune system. Moreover, public opinion is rather refractory to the use of adjuvants.
Finally, one strategy consists in optimizing the step of extracting the vaccine antigens (“split”) downstream of the production process: the objective being to obtain better and more extraction of the vaccine antigens while more effectively preserving their conformational structure and thus their antigenic property on which the protective power of the vaccine is directly dependent.
More specifically, the viral production obtained using infected embryonic hen eggs (harvesting of the allantoic fluid) or using culture of infected cell lines in a bioreactor (harvesting of the culture medium) is purified on a sucrose gradient and then inactivated by chemical treatment (preferentially with formalin, for obtaining according to the regulations a drop in the viral titer of 15 logs). The process subsequently involves the use of a detergent that will disrupt/fragment (“disrupting/fragmenting”) the viruses (“split virions”) and purification of the vaccine antigens by diafiltration.
The split, or fragmented virus, obtained is combined with partial or total solubilization of the viral proteins, thus modifying the (infectious) integrity and function of the viruses. Splitting also constitutes a method of inactivation supplementary to the chemical method; the term orthogonal inactivation is then used.
The split can be obtained by treating the purified viruses with various detergents, in particular with non-ionic and ionic (e.g. cationic) surfactants, such as: alkylglycosides, alkylthioglycosides, acyl sugars, sulfobetaines, betaines, polyoxymethylene alkyl ethers, N,N-dialkyl-glucamides, hecameg, alkylphenoxy-polyethoxyethanols, quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammonium bromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammonium salts, lipofectin, lipofectamine, DOT-MA (dodecyltrimethylammonium bromide), octyl- or nonylphenoxy polyoxyethanols (e.g. Triton X-100 or Triton NI 01), polyoxymethylene sorbitan esters (Tween surfactants), polyoxyethylene ethers, ethyl ether, polysorbate 80, deoxycholate, Tergitol® NP9, etc.
In this respect, numerous methods for splitting (fragmentation) of influenza viruses are known. Document WO 02/28422 ([8]) describes for example a splitting method by means of a splitting agent chosen from laureth 9, NaDOC, Sarcosyl group, Tween 80™ and Triton X100. Document WO 02/067983 ([9]) describes for its part a splitting method by means of sodium deoxycholate.
Surfactants (or detergents) are amphiphilic molecules composed of clearly distinct polar and nonpolar domains having a marked solubility in water. Beyond a precise concentration called the Critical Micelle Concentration (CMC), the micellization phenomenon is directed by the hydrophobic effect (M. Rosen, “Surfactants and Interfacial Phenomena”, 3rd Ed., Hoboken: John Wiley & Sons, Inc., 2004 ([10])).
The nature of the hydrophobic tail, its length, its degree of unsaturation and of branching, the presence or absence of aromatic nuclei and the number of chains affect the chemical properties of the surfactant monomers and their self-assembly in aqueous solution (CMC, aggregation number, size and nature of the geometric form of the aggregates). The nature of the hydrophilic group (neutral or charged, small or bulky) has a strong impact on the properties of the surfactant and its biological application.
Surfactants are categorized in three major families depending on the nature of their hydrophilic part (A. M. Schwartz, J. W. Perry, J. Berch, “Surface Active Agents and Detergents”, vol II, R. Krieger Pub. Co., New York, 1977 ([11])): ionic (cationic or anionic) surfactants, zwitterionic (or amphoteric) surfactants and neutral surfactants. They are often referenced as being either soft or harsh. It is possible to make an order of classification of detergents according to their harshest class to their softest class in this sense: ionic>amphoteric>neutral. Ionic surfactants, such as CTAB, are generally denaturing. They disrupt the intramolecular coulombic interactions of proteins and thus disorganize their three-dimensional conformation.
Zwitterionic surfactants (sulfobetaines, betaines) contain both a positive charge and a negative charge in their hydrophilic part and are electrically neutral.
Neutral surfactants, including in particular alkyl glucosides and polyoxymethylene alkyl ethers are characterized by non-charged hydrophilic heads. They are soft and non-denaturing surfactants which, however, disrupt protein-lipid and lipid-lipid interactions, and have no effect on the intramolecular coulombic interactions of proteins.
Detergents comprising a hydrophilic part of polyethylene glycol type (Triton X100, Brij®, Tween, etc.) are commonly used in biochemistry. They have the disadvantage of being considered to be molecules that are chemically heterogeneous with a variable polymerization index n on their PEG (polyethylene glycol) groups. These batches of commercial detergents are therefore not in the form of a species that is chemically well defined in solution, which can lead to a variation in their physico-chemical properties.
In the current context of the emergence of a pandemic pathogenic virus, an infection, for example a flu infection, could lead to 1.3 to 2 million hospitalizations and from 280 000 to 650 000 deaths in industrialized countries alone (WHO data).
One of the major economic challenges is thus to be able to reduce the cost of manufacturing a vaccine dose (more doses per production and/or reduction in the time for obtaining the same amount of doses).
In this context and from an economical point of view, the search for and development of new optimized processes for producing vaccine seeds are legitimate, in particular in terms (i) of improving production yields, (ii) of reducing time and/or costs, (iii) of producing antigens that are more immunogenic.